Infections by RNA or DNA viruses are a substantial threat to the health of humans and animals. To the RNA viruses belong the negative-strand-RNA viruses, such as for example, influenza viruses or the Borna disease virus. Infections by influenza viruses is the source of large-scale epidemics and cause a large number of fatalities on an annual basis. They are an immense cost factor in the economy, for instance by causing lost work days due to illness. Of substantial economic importance are also infections caused by the Borna disease virus (BDV), in particular those that attack horses and sheep, which have been isolated in man, too, and which have been connected with neurological diseases.
The problem of controlling in particular RNA viruses is the adaptability of the viruses caused by a high fault rate of the viral polymerases. Thus the preparation of suitable vaccines as well as the design of antiviral substances has been very difficult. Furthermore, it has been found that the application of antiviral substances immediately directed against the functions of the virus, has a relatively fair antiviral effect at the early stage of therapy, but will lead very quickly to the generation of resistant variants by mutation. An example is the anti-influenza drug amantadine and its derivatives, which is or are directed against a transmembrane protein of the virus. Within a short time after application, resistant variants of the virus are generated.
Other examples are the new therapeutic agents for influenza reactions that inhibit inhibiting the influenza-viral surface protein, neuraminidase. Hereto belongs, for instance, Relanza. In patients, Relanza-resistant variants have already been found (Gubareva et al., J Infect Dis 178, 1257-1262, 1998). The hopes placed on this therapeutic agent thus could not be fulfilled.
Due to their in most cases very small genomes and therefore limited coding capacity for replication-necessary functions, all viruses have to rely to a large extent on functions of their host cells. By exerting influence on such cellular functions necessary for viral replication, it is possible to negatively affect the virus replication in the infected cell. Then, there is no possibility for the virus to replace the missing cellular function by adaptation, in particular by mutation, in order to avoid the selection pressure. This could already be shown in the example of the influenza A virus with relatively unspecific inhibiting substances against cellular kinases and methyltransferases (Scholtissek and Müller, Arch Virol 119, 111-118, 1991).
It is known that cells have a multitude of signal transduction pathways, by means of which signals acting on the cell are transmitted to the cell nucleus. Thereby the cell is able to react to outside stimuli with cell proliferation, cell activation, differentiation or controlled cell death.
These signal transduction pathways have in common at least one kinase, which activates by phosphorylation at least one protein that transduces the signal.
By observing the cellular processes induced as a result of virus infections, it can be found that a multitude of DNA and RNA viruses activate in the infected host cell preferably by a defined signal transduction pathway, the so-called Raf/MEK/ERK kinase signal transduction pathway. This signal transduction pathway is one of the most important signal transduction pathways in a cell and plays a substantial role in proliferation and differentiation processes (Cohen, Trends in Cell Biol 7, 353-361, 1997; Robinson and Cobb, Curr. Opin. Cell Biol 9, 180-186, 1997; Treismann, Curr. Opin. Cell Biol 8, 205-215, 1996).
The investigation of the role of this signal transduction pathway in cellular decision processes has led to the identification of several pharmacological inhibitors inhibiting the signal transduction pathway, among other places, on the MEK level, i.e. relatively at the beginning of the signal transduction pathway (Alessi et al., J Biol Chem 270, 27489-27494, 1995; Cohen, Trends in Cell Biol 7, 353-361, 1997; Dudley et al., PNAS USA 92, 7686-7689, 1995; Favata et al., J Biol Chem 273, 18623-18632, 1998).
Newer data show that the inhibition of the Ras-Raf-MEK-ERK signal transduction pathway or of another signal transduction pathway, the MEKK/SEK/JNK signal transduction pathway, can drastically inhibit by active ingredients, which relatively selectively inhibit one of the kinases involved in this signal transduction pathway, for instance the MEK or the SEK, the intracellular multiplication of intranuclearly replicating negative-strand viruses, for instance of influenza A viruses and the Borna disease virus (BDV) (Pleschka et al., Nature Cell Biol 3, 301-305, 2001; Planz et al., J Virol 10, 4871-4877, 2001; PCT/DE 01/01292; DE 101 38 912).
Up to now it was known that influenza viruses preferably use the Raf-MEK-ERK signal transduction pathway or the MEKK/SEK signal transduction pathway for their multiplication, and that therefore an inhibition of these signal transduction pathways would lead to a complete inhibition of virus multiplication. Since, however, in a cell the signal transduction pathways have hardly any function closed in itself, but with activation of the one signal transduction pathway, further signal transduction pathways are additionally activated by cross linkages, it can in principle not be excluded that an inhibited signal transduction pathway can be bypassed by the cell as well as by the viruses, and the therapeutic effect of an active ingredient inhibiting a virus multiplication by inhibition of a certain signal transduction pathway could be limited thereby.
Therefore, there is a great need of identifying and using antiviral active ingredients, which act in addition to or as a supplement to those, which inhibit kinases of cellular signal transduction pathways, for instance of the Raf-MEK-ERK signal transduction pathway or of the MEKK/SEK/JNK signal transduction pathway. Such active ingredients have already been described in the documents PCT/DE 01/01292 and DE 101 38 912.
One of the most important signal transduction pathways in the cell is the nuclear factor of kappaB (NF-kB) signal transduction pathway. The central component of this transduction pathway is the heterodimer NF-kB transcription protein complex consisting on the one hand of p50 [formed by proteolysis of NF-kB1 (p105)] or of p52 [formed by proteolysis of NF-kappaB2 (p100)], and on the other hand of p65 (RELA), c-REL or RELB. The most common NF-kB complex is composed of p50 and p65.
The transcription activity of this NF-kB complex is inhibited by the binding of the inhibitor proteins of NFkB (IkB) to the nuclear binding sequence of p65. Thereby, the complex remains in the cytoplasm. An activation of the cell, for instance by growth factors, chemokines, TNF-alpha, Il-1, C40 ligand, LPS or by an infection by viruses will lead to the activation of kinases such as the “NF-kB inducing kinase” (NIK), the kinase TAK, the kinase AKT and possibly also of the kinase MEKK1. These kinases lead to the activation of the “inhibitor of kB (IkB) kinase” (IKK) complex, composed of IKKalpha, IKKbeta and NEMO. Activated IKK phosphorylates IkB and thus leads to its degradation and permits thereby the release, nuclear translocation and activation of the transcription activity of the p50/p65 heterodimer. Another NF-kB complex is composed of p100 and RELB. An activation of the cell by lymphotoxines will lead, probably preferably mediated by NIK, to the activation of IKKalpha. The activated IKK induces the phosphorylation and thus the proteolysis of p100 to p52, which in turn can translocate in the complex with the RELB into the cell nucleus and act there as a transcription factor.
It is known in the art that the activation of the NF-kB signal transduction pathway can be inhibited by i) nonsteroidal anti-inflammatory drugs (NSAIDs), such as sulindac (Yamamoto et al., J Biol Chem 274, 27307-27314, 1999; Berman et al., Clin Cancer Res 8, 354-360, 2002) or derivatives of sulindac such as sulindac sulphoxide, sulindac sulphone, sulindac sulphide or benzylamide sulindac analogues (Moon and Lerner, Cancer Research 62, 5711-5719, 2002) or acetylsalicylic acid or salicylic acid (Yin et al., Nature 396, 77-80, 1998) or curcumin (Oncogene 18, 6013-6020, 1999) by inhibition of the IKKbeta, ii) NEMO binding peptides (May et al., Science 289, 1550-1554, 2000), proteosome inhibitors such as PS-341 (Tan and Waldmann, Cancer Res 62, 1083-1086, 2002; Adams Trends Mol Med 8, 49-54, 2002), or iii) antisense nucleotide sequences specific for p65 or p50 (Higgins et al., PNAS-USA 90, 9901-9905, 1993). Up to now these inhibitors were, however, exclusively tested for their use as active ingredients for having influence on inflammation and the growth of tumors, since in both indications the involved cells have an increased activation of the NF-kB signal transduction pathway (Karin et al., Nature Reviews Cancer 2, 301-310, 2002).
To date, it has been postulated that a virus infection, in particular, by an influenza virus, activates the NF-kB signal pathway by the activation of the IKK, and said signal pathway is in turn decisively involved in the expression of antivirally active proteins increased by this infection, for instance interferon (Chu et al., Immunity 11, 721-731, 1999). In agreement with this idea is the finding that the influenza virus-induced activity of the interferon β promoter is strongly reduced in cells, which express a transdominant-negative mutation of IKK2 or IkBalpha (Wang et al., Virol 74, 11566-11573, 2000). On the other hand, there is a hint contradicting these ideas and findings that acetylsalicylic acid, known as an inhibitor of the IKK, is also able to inhibit influenza virus infections in the cell culture, however, such inhibition was observed only beginning from concentrations of 5-10 mM (corresponding to 0.9-1.8 mg/ml). Such concentrations are practically not achievable in blood without substantial side effects from oral administration of acetylsalicylic acid (Huang and Dietsch, New Engl J Med 319, 797, 1988). Acetylsalicylic acid is deemed the most toxic of all readily available analgesics with the smallest therapeutic width (Jones, Am J Ther 9, 245-257, 2002). Huang and Dietsch speculated that an administration by aerosol would result in higher and thus antivirally effective concentrations locally in the respiratory passages. These recommendations have, however, not been realized up to now, since the up to now known clinical side effects alone led, after administration of a dose of 100 mg acetylsalicylic acid otherwise not deemed toxic, to a warning not to take aspirin in case of a virus influenza, particularly by children (user information Z. No. 14.252 of Bayer AG about the acetylsalicylic acid (ASA) preparation Aspirin®).